1. Field of the Invention
The invention relates to a process for the fermentative production of full-length antibodies using Escherichia coli strains able to release proteins into the fermentation medium.
2. Background Art
The market for recombinant protein pharmaceuticals (biologics) has grown greatly in recent years. However, because the production costs for protein-based active pharmaceutical ingredients are still very high, there is a continual search for more efficient and more cost-effective processes and systems for producing them.
A particularly important class of proteins is antibodies. Antibodies are employed in research, in diagnosis and as therapeutic agent on a large scale, so that there is a need for production processes, which are particularly efficient and possible on the industrial scale.
In the case of antibodies, a distinction is made between full-length antibodies and antibody fragments. Full-length antibodies consist of four protein chains, two identical heavy chains and two identical light chains. The various chains are linked together by disulfide bridges. Each heavy chain is composed of a variable region (VH) and of a constant region, which includes the three domains CH1, CH2 and CH3. The region of the heavy chain which includes the CH2 and CH3 domains and which is also referred to as Fc region is not involved in antigen binding, but has other functions such as, for example, activation of the complement system. Each light chain is composed of a variable region (VL) and of a constant region, which includes the CL domain.
Antibodies (immunoglobulins) are assigned to five classes depending on the amino acid sequence of the heavy chain: IgA, IgD, IgE, IgG and IgM. The term full-length antibody means all antibodies in which the light chains in each case include the VL and CL domains, and the heavy chains are substantially composed of the VH-CH1-CH2-CH3 domains. Therefore, the antibody is able to carry out other functions (e.g. activation of the complement system), besides being able to bind a specific antigen.
By contrast, antibody fragments consist of only parts of a full-length antibody, normally the part including the antigen binding sites.
The organism most frequently used at present for producing recombinant proteins is the gram-negative enterobacterium Escherichia coli, because its genetics and physiology have been very well investigated, the generation time is short and manipulation is easy. This organism is likewise used to produce antibody fragments.
In contrast to antibody fragments, there have to date been only very few attempts to produce full-length antibodies in E. coli. Because of the size and complex structure of full-length antibodies, it is difficult to obtain correctly folded and assembled antibodies. Cytoplasmic production in E. coli is not possible in this case because E. coli does not form disulfide bridges in the cytoplasm. WO02/061090 describes the periplasmic production of full-length antibodies in E. coli. For this purpose, a specially designed expression vector on which the light chain and the heavy chain are expressed independently of one another in different promoter-cistron pairs is used. The two chains are in this case each fused to signal peptides and are transported by the usual Sec pathway in E. coli into the periplasm, where the folding and assembling takes place. To obtain the antibodies in this case it is necessary to disrupt the cells. The yields do not exceed 156 mg/l. Higher yields up to 880 mg/l were achieved only when periplasmic folding assistants (chaperones), such as the dsb proteins or FkpA, were coexpressed on plasmids in addition to the antibody chains. It is necessary to purify the antibodies from the large number of other E. coli proteins.
This process has the disadvantage for the industrial production of antibodies that the E. coli cells must be disrupted, and the target protein must be purified from the large number of other E. coli proteins. Any coexpression of periplasmic chaperones impedes this purification even further through the considerable additional amounts of unwanted proteins.